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Development of a universal system for fungal species identification and SNP typing via onchip minisequencing

: Mai, M.K.; Gfell, M.; Hauser, N.C.; Bauser, C.; Rohde, B.; Ferrari, S.; Coste, A.; Sanglard, D.; Bader, O.; Weig, M.; Groß, U.; Mellado, E.; Rupp, S.

Federation of European Biochemical Societies -FEBS-, Copenhagen; Federation of European Microbiological Societies -FEMS-:
3rd FEBS advanced lecture course Human Fungal Pathogens (HFP). Molecular mechanisms of host-pathogen interactions and virulence. Abstract book : May 2-8, 2009 La Colle sur Loup, France
La Colle sur Loup, 2009
FEBS advanced lecture course Human Fungal Pathogens (HFP) <3, 2009, La Colle sur Loup>
Fraunhofer IGB ()

Fungal infections are a predominant clinical problem, especially in intensive care units. In
particular for patients with a defective immune system, fungal infections are associated with high
mortality rates. Nevertheless, a fast and well directed medication may improve patient outcome
significantly. A further problem is the increasing resistance against the leading antimycotics due to
the rise of inherently resistant fungal species or during long term treatment. The resistance
mechanisms of fungal pathogens are often based on single nucleotide polymorphisms (SNPs) in
genes regulating the expression of pumps extruding the drug outside the fungal cell or encoding
for the target of the antimycotica. Since development of resistance is not predictable, constant
resistance monitoring is necessary to enable adequate patient medication. In the present study we
developed a system for the highly parallel detection of fungal species and their SNPs associated
with azole resistance using an on-chip minisequencing technology.
Minisequencing allows parallel analysis of SNPs both in homo- and heterozygous strains and
offers a good platform in terms of species identification. For the minisequencing reaction the
spotted probes are hybridized with PCR products from clinical samples and synthetic control
probes. In the enzymatic reaction, different fluorescently labelled dideoxynucleotides and a
thermo stable sequenase are used for the specific extension of the probes with the perfectly
matching nucleotide.
Based on this system, we developed a prototype chip with 15 species specific probes for Candida
albicans, C. glabrata and Aspergillus fumigatus based on ITS or 18s rRNA sequences as well as
SNP probes for erg11, tac1 and mrr1 SNPs of C. albicans from our existing SNP chip. Mutations
in these genes are central for causing resistance. Furthermore, four control probes used to confirm
the correct incorporation of the fluorescently labelled didesoxynucleotides have been developed to
enable normalisation, which is essential for correct identification of heterozygosity. The chip has
been successfully validated with synthetic templates and with defined PCR products from clinical
isolates. In the future, the chip will be expanded by further resistance associated SNPs and by onchip
minisequencing based species identification probes. This work has been financially supported
by the EURESFUN project (EU-FP6-STREP).